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《Journal of Clinical Rehabilitative Tissue Engineering Research》 2009-28
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Package of recombinant adeno-associated virus encoding cell division cycle 2-siRNA

Wei Jia-jun,Wang Zhuo-ran Department of Neurology,Renmin Hospital of Wuhan University,Wuhan 430060,Hubei Province,China  
BACKGROUND:Cell division cycle 2 (cdc2) plays an important role in the course of neuronal degeneration in neurodegenerative diseases such as Alzheimer's disease. The silencing of cdc2 gene with adeno-associated virus vector might protect the neurons in neurodegenerative diseases. OBJECTIVE:To pack recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. DESIGN,TIME AND SETTING:Blank control experiment was performed in the laboratory of Department of Neurology at Wuhan Tongji Hospital between October 2007 and August 2008. MATERIALS:Helper Free adeno-associated virus system (pAAV-MCS-EGFP,p-RC,p-Helper) and AAV-293 cells were purchased from Stratagene. METHODS:The pAAV-MCS-U6-cdc2-siRNA-EGFP expressing plasmid was constructed by molecular biological techniques. The cotransfection of this plasmid,together with p-RC and p-Helper into AAV-293 cells was mediated by calcium acid phosphate. The rAAV encoding cdc2-siRNA (rAAV-U6-cdc2-siRNA-EGFP) was packed and harvested. The silencing effect of this virus on cdc2 gene expression in AAV-293 cells was assessed by Western Blot,and its titer was determined by spot hybridization method. MAIN OUTCOME MEASURES:sequencing of pAAV-MCS-U6-cdc2-siRNA-EGFP plasmid inserting U6-cdc2-siRNA; 3 plasmid pAAV-MCS-U6-cdc2-siRNA-EGFP,p-RC,p-Helper cotransfecting AAV-293 cells; cdc2 expression after rAAV-U6-cdc2-siRNA-EGFP infected AAV-293 cells. RESULTS:① The successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP plasmid was proved by DNA sequencing. ② Green fluorescent protein was expressed in AAV-293 cells,and the contransfection was working well. ③ The cdc2 gene expression in AAV-293 cells was down-regulated markedly after infection of rAAV-U6-cdc2-siRNA-EGFP. ④ The titer of rAAV-U6-cdc2-siRNA-EGFP was 1x1012 v.g/mL. CONCLUSION:The package of rAAV encoding cdc2-siRNA is successful. It can silence cdc2 gene effectively.
【CateGory Index】: R346
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